Vitamin D acts on the intestine through a mechanism that is steroid hormone-like, to increase calcium transport. The interaction of 1,25-(OH)2-D3 with its specific receptor protein in intestinal mucosal cells regulates a gene coding for vitamin D-dependent calcium-binding protein (CaBP) which is thought to function in the transport process. The recent discovery of putative 1,25-(OH)2-D3 receptors and CaBP in kidney, bone, brain and other tissues prompted the concept that vitamin D may function in these organs through an effector system with common components. The hypothesis, while attractive, has not been rigorously tested. There are significant outstanding questions regarding the exact cellular locations of 1,25-(OH)2-D3 receptors and CaBP and it remains unclear as to whether the synthesis of CaBP in non-intestinal tissue is regulated directly by 1,25-(OH)2-D3. The goal is to gain answers to these questions by bringing to bear new powerful techniques not previously available. First, newly developed immunocytochemical techniques will be used to undertake the first simultaneous mapping of the 1,25-(OH)2-D3 receptor and CaBP in kidney, bone and brain of the adult and developing rat. With such an approach we will identify exactly which cells within each tissue are likely to be responsive to vitamin D. Both immunocytochemical methods and specific radioimmunoassays will then be employed to determine the relative importance of vitamin D deficiency and hypocalcemia in the regulation of CaBP synthesis in these non-intestinal tissues. A final aspect of this project will attempt to probe more directly, the mechanism by which the synthesis of renal CaBP is regulated. In these studies, primary kidney epithelial cells that express CaBP and retain the ability to respond to vitamin D will be used to evaluate the effects of 1,25-(OH)2-D3 and intracellular calcium ion concentration on CaBP synthesis in vitro. The results obtained from these studies will promote a clearer understanding of the relationship between vitamin D and CaBP and thus provide a strong foundation for future studies of possible functions for vitamin D in these tissues.